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Blocking and detection chemistries affect antibody performance on reverse phase protein arrays
Author(s) -
Ambroz Kristi L. H.,
Zhang Yonghong,
SchutzGeschwender Amy,
Olive D. Michael
Publication year - 2008
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.200700676
Subject(s) - blocking (statistics) , blot , antibody , chemistry , microbiology and biotechnology , biology , biochemistry , immunology , computer science , gene , computer network
Antibody specificity is critical for RP protein arrays (RPA). The effects of blocking and detection chemistries on antibody specificity were evaluated for Western blots and RPA. Blocking buffers significantly affected nonspecific banding on Western blots, with corresponding effects on arrays. Tyramide signal amplification (TSA) increased both specific and nonspecific signals on Westerns and arrays, masking the expected gradations in signal intensity. These results suggest that consistent blocking and detection conditions should be used for antibody validation and subsequent RPA experiments.